Femtotips 1

All microinjection experiments were performed using a Femtojet and Injectman NI2 (Eppendorf) microinjection system fitted with Femtotips I (Eppendorf). Cells were incubated in DMEM with no phenol red, supplemented with 10% FBS, in glass bottom dishes (Mattek) for all injection experiments.
In addition to microporation and microinjection, we anticipate that other cellular delivery methods previously used in MB research, including lipofectamine, nanoparticles, TAT-peptide, and streptolysin-O^{13 (link), 16 (link)–22 (link)}, should be capable of delivering the MBs used in this study as well.

Escherichia coli and M. luteus were cultured to OD₆₀₀ 0.7, pelleted, washed and resuspended in PBS. The final OD₆₀₀ for infection was 0.4 for E. coli and 0.1 for M. luteus. Four days after the dsRNA injection, mosquitoes were infected with the bacteria by inserting Femtotips® I (Eppendorf) microcapillary in the bacteria culture and poking it in the mosquito’s thorax. The adult mosquitoes that did not recover after 3 h of being exposed to the bacteria were not considered for the analysis. Dead mosquitoes were counted daily and recorded for 10 days. At least three independent experiments were performed, each with at least 25 female mosquitoes per group. The survival analysis was performed using Kaplan-Meier log-rank test.

The sequence obtained from RACE was used to construct double-stranded RNA using the T7 Megascript Kit (Ambion, Foster City, USA). A 445 bp Rel2 region was amplified and the amplicon was ligated downstream of the T7 promoter in the pGEM®-T Vector (Promega) to produce sense and antisense RNA strands. Approximately 50–200 nl of dsRNA (2 μg/μl) was injected into the thorax of anesthetized 2–4 days old female mosquitoes by using InjectMan® NI 2 (Eppendorf, Hamburg, Germany) and FemtoJet® express (Eppendorf) with Femtotips® I (Eppendorf) microcapillaries. The dsLacZ (441 bp) was used in the control injection. The primers used for the construction of dsRel2 and dsLacZ are shown in Table 1.